Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A nucleotide sequence of tRNA(Tyr) from the extreme thermophile Thermus thermophilus HB-27 living at 75 degrees C was determined. It is 86 nt long and shares a 52% homology with tRNA(Tyr) from Escherichia coli. A comparative analysis of the interaction sites of tRNA(Tyr) from T. thermophilus and E. coli with the cognate aminoacyl-tRNA synthetases was accomplished by the chemical modification and nuclease hydrolysis approaches. The tRNA(Tyr) was shown to interact with the cognate enzyme in the anticodon stem (on the 5'-side), in the anticodon, in the variable stem and loop (on the 5'-side), and in the acceptor stem (on the 3'-side). These regions are located in the variable stem of the L-form. It was demonstrated that, upon forming the complex E. coli tRNA(Tyr)-cognate synthetase, endonuclease V1 induces additional cleavages of phosphodiester bonds on the 3'-side of the anticodon stem and on the 5'-side of the T-stem. This implies that tRNA may change its conformation when it interacts with the enzyme.
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PMID:[Comparative analysis of interaction sites of Thermus thermophilus and Escherichia coli tRNA(Tyr) with homologous aminoacyl-tRNA synthetases by means of chemical modification and nuclease hydrolysis]. 978 79

To improve the production of D-amino acids using an immobilized N-carbamyl-D-amino acid amidohydrolase, the enzyme gene of Agrobacterium sp. KNK712 was mutagenized randomly to increase its thermostability. The gene was inserted into M13mp19, mutagenized with hydroxylamine, ligated into pUC19 after restriction endonuclease digestion, and then used to transform Escherichia coli. The resultant transformants were screened by a newly developed colorimetric enzyme assay method, and the candidate colonies corresponding to red spots were separated from the master plates. Using cell-free extracts of these clones, the properties of the enzymes produced were investigated, it being proved that these enzymes had almost the same activity and improved thermostability by about 5 degrees C compared with those of the native enzyme. As found on enzyme gene analysis of these mutants, the 57th amino acid, histidine, of the enzyme was changed to tyrosine, or the 203rd amino acid, proline, to leucine or serine.
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PMID:Increase in thermostability of N-carbamyl-D-amino acid amidohydrolase on amino acid substitutions. 980 66

Ribonuclease P activity from infusoria Tetrahymena pyriformis has been isolated and purified more than 1000-fold over cytosol crude extract. Purified tRNA 5' endonuclease processes in vitro heterologous substrates, precursors of the human tRNA(Tyr) and Drosophila melanogaster tRNA(Leu), exactly at the 5' end of the mature molecules. The activity was abolished by micrococcal nuclease and protease treatment indicating that both RNA and protein components are essential for its activity. The most abundant polypeptides in the purified enzyme fractions have molecular masses of about 100, 44 and 35 kDa. The enzyme requires divalent cations for its activity and shows optimal activity in the presence of the low concentrations of the monovalent salts. Substrate structural requirements for the purified enzyme were analyzed with different tRNA precursor models. The analysis of the derivatives of tRNA(Leu) precursors with altered aminoacyl stem structures reveals that end of the stem is important for substrate 5' end processing with purified enzyme.
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PMID:Partial characterization of the ribonuclease P from Tetrahymena pyriformis. 981 Apr 66

The amino acid residue Asn141 of the restriction endonuclease EcoRI was proposed to make three hydrogen bonds to both adenine residues within the recognition sequence -GAATTC-. We have mutated Asn141 to alanine, aspartate, serine, and tyrosine. Only the serine mutant is active under normal buffer conditions although 1000-fold less than wild-type EcoRI. The alanine and aspartate mutants can be activated by Mn2+. At acidic pH the latter mutant becomes even more active than the wild-type enzyme in the presence of Mn2+. We conclude that Asn141 is essential for DNA recognition and that serine can partly substitute it.
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PMID:Asn141 is essential for DNA recognition by EcoRI restriction endonuclease. 982 60

During the course of work aimed at isolating a rice gene from Oryza australiensis by PCR, the oligonucleotide primers used were found to generate a fragment that showed sequence homology to the endonuclease (EN) region of the maize non-LTR retrotransposon (LINE) Cin4. We carried out further PCRs using oligonucleotide primers that hybridized to these sequences, and found that they amplified several fragments, each with homology to the EN regions, from Oryza sativa cv. Nipponbare as well as O. australiensis. We mapped the approximate locations of two rice LINE homologues by screening clones in a YAC library made from a rice (O. sativa) genome, and found that each homologue was present in a low copy number apparently at nonspecific regions on rice chromosomes. We then carried out PCR using degenerate oligonucleotide primers which hybridized to the rice LINE homologues and Cin4 to ascertain whether LINE homologues are present in a variety of members of the plant kingdom, including angiosperms, gymnosperms, bracken, horsetail and liverwort. Cloning and nucleotide sequencing revealed that 53 clones obtained from 27 out of 33 plant species contained LINE homologues. In addition to these homologues, we identified four homologues with EN regions in the Arabidopsis thaliana genome by a computer search of databases. The nucleotide sequences of almost all the LINE homologues were greatly diverged, but the derived amino acid sequences were well conserved, and all contained glutamic acid and tyrosine residues at almost the same relative positions as in the the active site regions of AP (apurinic/apyrimidinic)-endonucleases. The EN regions in the LINE homologues from closely related plant species show a closer phylogenetic relationship, indicating that sequence divergence during vertical transmission has been a major influence upon the evolution of plant LINEs.
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PMID:Non-LTR retrotransposons (LINEs) as ubiquitous components of plant genomes. 1007 Dec 12

The activities of restriction enzymes are important examples of Mg(II)-dependent hydrolysis of DNA. While a number of crystallographic studies of enzyme-DNA complexes have also involved metal ions, there have been no solution studies exploring the relationship between enzyme conformation and metal-ion binding in restriction enzymes. Using PvuII restriction endonuclease as a model system, we have successfully developed biosynthetic fluorination and NMR spectroscopy as a solution probe of restriction-enzyme conformation. The utility of this method is demonstrated with a study of metal-ion binding by PvuII endonuclease. Replacement of 74% (+/- 10%) of the Tyr residues in PvuII endonuclease by 3-fluorotyrosine produces an enzyme with Mg(II)-supported specific activity and sequence specificity that is indistinguishable from that of the native enzyme. Mn(II) supports residual activity of both the native and fluorinated enzymes; Ca(II) does not support activity in either enzyme, a result consistent with previous studies. 1H- and 19F-NMR spectroscopic studies reveal that while Mg(II) does not alter the enzyme conformation, the paramagnetic Mn(II) produces both short-range spectral broadening and longer range changes in chemical shift. Most interestingly, Ca(II) binding perturbs a larger number of different resonances than Mn(II). Coupled with earlier mutagenesis studies that place Ca(II) in the active site [Nastri, H. G., Evans, P.D., Walker, I.H. & Riggs, P.D. (1997) J. Biol. Chem. 272, 25761-25767], these data suggest that the enzyme makes conformational adjustments to accommodate the distinct geometric preferences of Ca(II) and may play a role in the inability of this metal ion to support activity in restriction enzymes.
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PMID:Effects of divalent metal ions on the activity and conformation of native and 3-fluorotyrosine-PvuII endonucleases. 1010 58

N-Nitrosodimethylamine (NDMA) is an acute hepatotoxin and potent carcinogen. The metabolic activation of NDMA to reactive metabolites is a critical step for the expression of its toxic and carcinogenic potential. We have previously demonstrated a strong correlation between methylation of cellular macromolecules and NDMA-mediated cytotoxicity, and we have demonstrated that reactive oxygen species may partially contribute to the toxic effects in P450 2E1-expressing cells. The mode of cell death in NDMA-treated monolayer cultures exhibited the following characteristics: (i) condensation of nuclear chromatin as demonstrated by using Hoechst 33258 staining, (ii) DNA fragmentation as detected by combining pulsed field and conventional agarose gel electrophoresis, and (iii) DNA double strand breaks determined by using the in situ terminal deoxynucleotidyl transferase assay and flow cytometric analysis. These results indicate that reactive metabolites of NDMA trigger activation of the signal pathway for apoptotic cell death in these P450-expressing cells. The NDMA-mediated cell death was partially prevented by the endonuclease inhibitor, aurintricarboxylic acid, as well as the caspase inhibitors, acetyl-Asp-Glu-Val-Asp-CHO and acetyl-Tyr-Val-Ala-Asp-CHO. The cell cycle distribution was altered in NDMA-treated cells resulting in an increase in the G2/M phase and a decrease in the G1 phase. Our results suggest that DNA degradation, the inability to complete DNA repair, the biochemical events associated with G2/M arrest, and the process of apoptotic death all result from P450 2E1-catalyzed metabolism of NDMA.
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PMID:N-Nitrosodimethylamine-mediated cytotoxicity in a cell line expressing P450 2E1: evidence for apoptotic cell death. 1036 44

The endonuclease activity of the Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) cuts at the terminal resolution site (trs) within the hairpin structure formed by the AAV inverted terminal repeats. Recent studies suggest that a DNA unwinding function of Rep68/78 may be required for endonuclease activity. We demonstrate that several mutant proteins which are endonuclease negative on a fully duplex hairpin substrate are endonuclease positive on a partially single-stranded hairpin substrate. Truncation analysis revealed that the endonuclease function is contained within the first 200 amino acids of Rep68/78. This endonucleolytic cleavage is believed to involve the covalent attachment of Rep68/78 to the trs via a phosphate-tyrosine linkage. A previous report (S. L. Walker, R. S. Wonderling, and R. A. Owens, J. Virol. 71:2722-2730, 1997) suggested that tyrosine 152 was part of the active site. We individually mutated each tyrosine within the first 200 amino acids of the Rep68 moiety of a maltose binding protein-Rep68/78 fusion protein to phenylalanine. Only mutation of tyrosine 156 resulted in a protein incapable of covalent attachment to a partially single-stranded hairpin substrate, suggesting that tyrosine 156 is part of the endonuclease active site.
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PMID:Mutational analysis of adeno-associated virus type 2 Rep68 protein endonuclease activity on partially single-stranded substrates. 1068 15

An efficient method was devised to isolate temperature sensitive mutants of E. coli defective in tRNA biosynthesis. Mutants were selected for their inability to express suppressor activity after su3(+)-transducing phage infection. In virtually all the mutants tested, temperature sensitive synthesis of tRNA(Tyr) was demonstrated. Electrophoretic fractionation of (32)P labeled RNA synthesized at high temperature showed in some mutants changes in mobility of the main tRNA band and the appearance of slow migrating new species of RNA. Temperature sensitive function of mutant cells was also evident in tRNA synthes: directed by virulent phage T4 and BF23. We conclude that although the mutants show individual differences, many are temperature sensitive in tRNA maturation functions. In spite of much information on the structure and function of transfer RNA (tRNA), our knowledge concerning the biosynthesis of tRNA is relatively poor. It is generally assumed that complete tRNA molecules are made via a series of processing steps from the original transcription products of tRNA genes which are presumably unmodified and longer than mature tRNA molecules. In the case of tyrosine suppressor tRNA of su3(+), an unmodified precursor RNA carrying additional residues at the 3' and 5' ends has been isolated (1,2), and an endonuclease cleaving at the 5' side of this precursor has been identified in E. coli (3). In the case of T4 encoded tRNA, a large precursor molecule for several tRNA's has been reported (4). Some enzymes that catalyze the modifications have also been described (5). However, the over-all picture and the precise mechanisms of tRNA maturation are as yet largely unkown. For study of tRNA biosynthesis in E. coli, a genetic approach may prove useful, as has been the case in other biosynthetic pathways. In order to obtain mutants blocked in any of the intermediary steps of tRNA synthesis, we have developed an efficient selection system that enriches these mutants. Since any mutational block in tRNA biosynthesis might well be lethal, we looked for conditional lethal mutants in which the defect in tRNA synthesis occurs only at high temperature. In this selection system, the su3 gene carried by a temperate phage was newly introduced into cells(su(-)) and those cells incapable of synthesizing su3(+) tRNA at high temperature were selected. Such mutants were easily enriched by using conditions in which cells expressing suppressor activity were killed by two virulent phages. In this communication, we report the method for isolation of mutants and some characterization of tRNA synthesis in these mutants. Recently, Schedl and Primakoff (6) have independently isolated thermosensitive mutants of E. coli defective in tRNA synthesis which may or may not be different types from ours.
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PMID:Temperature sensitive mutants of Escherichia coli for tRNA synthesis. 1079 71

Thermal denaturation studies show that 10-15% of the calf thymus DNA in the heat denatured (Tyr-Gly-Tyr-Gly-Tyr)-DNA complex renatures spontaneously after colling. The double-strandness of this DNA was verified by its resistance to single-strand Neurospora endonuclease and by its elution profile on hydroxypatite columns. The renatured DNA isolated by the latter technique was found to contain 56% GC compared to the 41% GC content of the whole thymus DNA. Alternating tryptophanyl-glycyl and histidyl-glycyl peptides also catalyze the same renaturation. A linear correlation was found between the thermal stabilization afforded to the DNA by the various peptides and their ability to "catalyze" DNA strand renaturation.
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PMID:'DNA snapback' peptides. 1079 54


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